Bottom Line:
As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules.Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly.We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

ABSTRACTIpl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

fig6: Ipl1p's substrates localize to the spindle midzone, and Ipl1p follows the plus ends of the depolymerizing spindle. (A) Microscopy was performed on strains containing endogenous Ndc10–GFP (SBY539), Sli15–GFP (SBY875), or Dam1–GFP (SBY1115). The fluorescence images show that all three Ipl1p substrates localize to the spindle midzone. The corresponding DIC pictures are shown (right). (B) Live image analysis was performed on cells expressing Ipl1–GFP and Tub1–CFP (SBY1036). Every 30 s, five z sections at 0.5-μm intervals were acquired while alternating between the two channels (FITC and CFP). The deconvolved video shows tubulin in red, Ipl1p in green, and the overlapping signal in yellow. Before spindle disassembly, Ipl1p localizes to the spindle midzone (0'). When the spindle starts breaking down (1'), the Ipl1p signal splits and then follows the plus ends of the depolymerizing spindle (2') until it reaches the spindle poles (3.5′). Video 3 showing Ipl1p localization is available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1. Bars, 10 μm.

Mentions:
The novel midzone localization pattern for Ipl1p led us to test whether proteins that Ipl1p regulates also localize to the spindle midzone. First, we tested Cse4p which we have shown here is an Ipl1p substrate in vitro. Localization of a Cse4–GFP fusion showed that it does not transfer to the spindle (unpublished data). We next analyzed the localization of COOH-terminal GFP fusions to the Ndc10, Sli15, and Dam1 proteins (Fig. 6 A). Ndc10–GFP localized to the midzone in late anaphase cells in addition to the previously reported spindle and kinetochore localization (Lechner and Carbon, 1991; Goh and Kilmartin, 1993). We found that Sli15–GFP also accumulates at the spindle midzone and exhibits the same localization pattern as Ipl1p throughout the entire cell cycle (Fig. 6 A; unpublished data). Various labs have reported that Dam1p localizes to kinetochores throughout the cell cycle and to the mitotic spindle (Hofmann et al., 1998; He et al., 2001; Jones et al., 2001). Here we show that Dam1–GFP also localizes to the spindle midzone in anaphase cells. Therefore, the majority of known Ipl1p substrates localize to the spindle midzone, providing several potential candidates for Ipl1p regulation of spindle disassembly.

fig6: Ipl1p's substrates localize to the spindle midzone, and Ipl1p follows the plus ends of the depolymerizing spindle. (A) Microscopy was performed on strains containing endogenous Ndc10–GFP (SBY539), Sli15–GFP (SBY875), or Dam1–GFP (SBY1115). The fluorescence images show that all three Ipl1p substrates localize to the spindle midzone. The corresponding DIC pictures are shown (right). (B) Live image analysis was performed on cells expressing Ipl1–GFP and Tub1–CFP (SBY1036). Every 30 s, five z sections at 0.5-μm intervals were acquired while alternating between the two channels (FITC and CFP). The deconvolved video shows tubulin in red, Ipl1p in green, and the overlapping signal in yellow. Before spindle disassembly, Ipl1p localizes to the spindle midzone (0'). When the spindle starts breaking down (1'), the Ipl1p signal splits and then follows the plus ends of the depolymerizing spindle (2') until it reaches the spindle poles (3.5′). Video 3 showing Ipl1p localization is available at http://www.jcb.org/cgi/content/full/jcb.200209018/DC1. Bars, 10 μm.

Mentions:
The novel midzone localization pattern for Ipl1p led us to test whether proteins that Ipl1p regulates also localize to the spindle midzone. First, we tested Cse4p which we have shown here is an Ipl1p substrate in vitro. Localization of a Cse4–GFP fusion showed that it does not transfer to the spindle (unpublished data). We next analyzed the localization of COOH-terminal GFP fusions to the Ndc10, Sli15, and Dam1 proteins (Fig. 6 A). Ndc10–GFP localized to the midzone in late anaphase cells in addition to the previously reported spindle and kinetochore localization (Lechner and Carbon, 1991; Goh and Kilmartin, 1993). We found that Sli15–GFP also accumulates at the spindle midzone and exhibits the same localization pattern as Ipl1p throughout the entire cell cycle (Fig. 6 A; unpublished data). Various labs have reported that Dam1p localizes to kinetochores throughout the cell cycle and to the mitotic spindle (Hofmann et al., 1998; He et al., 2001; Jones et al., 2001). Here we show that Dam1–GFP also localizes to the spindle midzone in anaphase cells. Therefore, the majority of known Ipl1p substrates localize to the spindle midzone, providing several potential candidates for Ipl1p regulation of spindle disassembly.

Bottom Line:
As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules.Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly.We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.

ABSTRACTIpl1p is the budding yeast member of the Aurora family of protein kinases, critical regulators of genomic stability that are required for chromosome segregation, the spindle checkpoint, and cytokinesis. Using time-lapse microscopy, we found that Ipl1p also has a function in mitotic spindle disassembly that is separable from its previously identified roles. Ipl1-GFP localizes to kinetochores from G1 to metaphase, transfers to the spindle after metaphase, and accumulates at the spindle midzone late in anaphase. Ipl1p kinase activity increases at anaphase, and ipl1 mutants can stabilize fragile spindles. As the spindle disassembles, Ipl1p follows the plus ends of the depolymerizing spindle microtubules. Many Ipl1p substrates colocalize with Ipl1p to the spindle midzone, identifying additional proteins that may regulate spindle disassembly. We propose that Ipl1p regulates both the kinetochore and interpolar microtubule plus ends to regulate its various mitotic functions.